In an enzyme electrode assay method, a chemical substance of interest is selectively measured making use of the molecule-recognizing capacity of respective enzyme, generally using an apparatus comprising an enzyme-immobilized membrane and an electrode. In the field of biochemical assay methods, simple and quick measuring methods have been developed making use of enzyme sensors and the like and have already been disclosed for example in JP-B 02-59424 (the term "JP-B as used herein means an "examined Japanese patent publication), JP-A 60-17346 (the term "JP-A" as used herein means an "unexamined published Japanese patent application) and JP-A 60-17347.
There are a number of known specific binding assay methods such as an immunoassay in which an antigen-antibody reaction is employed, a receptor assay in which a receptor is used and a nucleic acid probe assay in which hybridization of complementary nucleic acid sequences is employed. Because of the high specificity, these assay methods are used frequently in various fields including clinical chemistry and the like.
On the other hand, many attempts have been made to apply electrochemical sensors to the specific binding assay method. For example, JP-A 58-58467 discloses a specific binding reaction on the surface of an electrode to which an enzyme (catalase)-labeled antibody preparation is applied, JP-A 2-179461 discloses a competitive specific binding reaction on the surface of an electrode to which an enzyme (glucose oxidase)-labeled substance is applied and JP-A 60-17360 discloses a specific binding reaction which is effected by the use of an oxidation-reduction enzyme (glucose oxidase or the like) and an electron mediator (a ferrocene derivative or the like).
Similar techniques have also been disclosed for example in JP-A 3-25360, JP-A 60-127450, JP-A 60-242361, JP-A 63-139248, JP-W 61-500706 (the term "JP-W" as used herein means an "unexamined published Japanese translation of PCT international patent application"), U.S. Pat. Nos. 4,963,245, 5,066,372, Anal. Chem., vol. 56, pp. 2355-2360 (1984) and Clin. Chem., vol. 31, pp. 1449-1452 (1985).
Of these enzyme electrode methods and specific binding assay methods, an amperometric enzyme electrode method, an electrochemical sensor-aided specific binding assay method or the like generally uses a low molecular weight compound, so-called electron mediator, which mediates between a biological oxidation-reduction reaction such as of an enzyme and an electrode reaction. In other words, the electron mediator is a compound which mediates electron transfer between an enzyme reaction and an electrode reaction in an assay system that comprises at least one oxidation-reduction enzyme and an electrode capable of transferring electrons in the system.
Such an electron mediator is "reduced/oxidized" by the enzyme reaction and "oxidized/reduced" by the electrode reaction. By circulating between these two reactions, it mediates transfer of electrons from the enzyme reaction to the electrode reaction or from the electrode reaction to the enzyme reaction.
Because of this, such an electron mediator is required to be a compound having certain characteristics such as
(1) it is efficiently oxidized or reduced by the enzyme reaction, PA1 (2) it is oxidized or reduced by the electrode reaction and then returned to a state which can be used by the enzyme reaction, and PA1 (3) it is stable when oxidized or reduced by the enzyme or electrode reaction. PA1 (1) low water solubility of oxidized or reduced type of the compound to be used as the electron mediator, PA1 (2) poor stability after drying of the compound to be used as the electron mediator because of its high vapor pressure, and PA1 (3) poor stability in biological sample such as blood or urine of oxidized type (or radical state) or reduced type of the compound to be used as the electron mediator.
In addition, the electron mediator which mediates electron transfer between the enzyme reactions and electrode reactions should be oxidized or reduced at an electric potential within a measurable range of the working electrode. The measurable range of electrode varies depending on the types of the electrode and solution conditions. In the case of a carbon electrode, it may be generally from -1.2 V to +1.0 V (vs. SCE).
There are a large number of such electron mediators known in the art including organic metal compounds such as ferrocene derivatives and the like (British Patent 8132034), inorganic compounds such as potassium ferri(or ferro)cyanide and the like and organic compounds such as p-phenylenediamine (PPD) derivatives and the like.
For example, it is known that a p-phenylenediamine (PPD) derivative, N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), mediates electron transfer between an electrode and sulfite oxidase which catalyzes oxidation of sulfite ions into sulfate ions (Anal. Chem., vol. 65, pp. 242-246, 1993). Also, WO 92/07263 discloses a benzene derivative having amino group, an alkylamino group, a dialkylamino group, a morpholinyl group or a piperidyl group at the 1,4-position, as an electron mediator whose reduced type is slightly soluble in water.
On the other hand, N,N,N',N'-tetrakis-(2'-hydroxyethyl)-p-phenylenediamine (THEPD) and N,N,N',N'-tetrakiscarboxymethyl-p-phenylenediamine (TCPD) as other PPD derivatives have been used as photo-sensitive materials and chelating agents (Photographic Science and Engineering, vol. 6, no. 1, p. 39, 1962; Nature, vol. 204, p. 180, 1964; Z. Chem., vol. 4, no. 11, p. 436, 1964; Z. Chem., vol. 4, no. 12, p. 463, 1964; An. Quin. Ser. B, vol. 59, no. 7-8, p. 501, 1963; An. Quin. Ser. B, vol. 61, no. 5, p. 717, 1965; An. Quin. Ser. B, vol. 61, no. 5, p. 723, 1965; An. Quin. Ser. B, vol. 59, no. 7-8, p. 493, 1963; An. Quin. Ser. B, vol. 82, no. 2, p. 133, 1986; Research Disclosure, vol. 212, p. 428, 1981). Also, it is known that THEPD can be used as a coloring reagent of a peroxidase reaction, because its reaction in the presence of peroxidase, a naphthol derivative and hydrogen peroxide results in the formation of a color substance due to binding of its free radical to the naphthol derivative (EP 0504663). It has not been known however that THEPD and TCPD can function as electron mediators which have the above mentioned features.
In this connection, since principal use of the prior art electron mediators in electron mediator-aided assay methods is enzyme electrodes, preferred electron mediators are those which are insoluble or slightly soluble in water as described in the aforementioned WO 92/07263. That is, in order to obtain a stable electrochemical signal through mediation of electron transfer between an electrode and an oxidation-reduction enzyme immobilized or insolubilized on the electrode, it is necessary to prevent effusion of the electron mediator into blulk solution of test sample, by allowing the mediator to exist close to the electrode.
Also, when the electron mediator is a compound which is apt to undergo influence of interfering substances contained in test samples, such as ascorbic acid, uric acid, hemoglobin, oxygen and the like, it is necessary to avoid its contact with the interfering substances by converting the electron mediator into a water-insoluble form and including it inside the electrode. However, since the electron mediator is included in the electrode, this method has disadvantages in that the field of reaction is limited, only an enzyme capable of contacting efficiently with the electron mediator included inside the electrode can participate in the reaction and the signal strength generally becomes weak.
Recently, disposable type enzyme electrode chips in which a water soluble electron mediator such as potassium ferricyanide or the like is used have been developed (cf. JP-A 1-156658). Since such a water soluble electron mediator can mediate transfer of electrons between an electrode and an enzyme located at a certain distant from the electrode, effective number of enzyme molecules increases and improvement of characteristics of the enzyme electrode becomes possible.
In the case of such disposable type enzyme electrode chips, it is desirable to incorporate necessary components for the function of the electrode into the chips in advance so that the measurement can be made by simply adding a test sample to the assay system. Because of this, it is desirable that such an electron mediator has a high solubility in water, can be dried easily and is stable under dry state. Water soluble electron mediators of metal complexes such as a ferrocene derivative, potassium ferricyanide, an osmium complex and the like can satisfy these conditions to some degree. However, being small in their transfer rate of electrons to or from the enzyme, it is necessary to use the electron mediators in a large quantity. In addition, since these water soluble electron mediators are reacted in test sample solutions, they are apt to undergo influence of interfering substances contained in test samples, such as ascorbic acid, uric acid, hemoglobin, oxygen and the like. Because of this, a compound which undergoes smaller influence of such interfering substances is expected to be developed.
In addition to the above, a specific binding assay method, especially, Mediator Diffusion-Controlled Immunoassay (being abbreviated MEDIA assay method hereinafter) has recently been developed in which, an oxidation-reduction enzyme is used as a labeling substance, and the labeling substances are developed in a matrix with a liquid sample to form distributions having the different distances from each labeling substance to the electrode through a specific binding reaction of an analyte in the liquid sample with a specific binding substance. The signal substances or electron mediators therefrom diffuse to the electrode and the diffusions are rate-determining step in this assay method. The currents of the signal substances or electron mediators are measured at the electrode to determine concentration of the analyte in the liquid sample. The distance distribution can be detected by the current, and the current is corresponding to the concentration of the analyte in the sample (cf. JP-A 5-264552 (EP 0 525 723 A2)). Such a type of assay method also requires development of an electron mediator which can mediate stable transfer of electrons between the enzyme and the electrode in liquid samples such as blood, urine and the like that contain interfering substances.
Thus, the present invention contemplates overcoming the aforementioned problems involved in the prior art, thereby providing an electrochemical assay method in which a water soluble electron mediator is used, such as an enzyme electrode method or a specific binding assay method, for example MEDIA method, which has excellent detection sensitivity (responsibility), can perform the measurement stably with high reproducibility even in the case of blood, urine and the like samples that contain interfering substances and can be applied suitably to dry chemistry use, as well as a novel p-phenylenediamine compound which is used as an electron mediator in the assay method.
In an assay method in which an electron mediator is used, more particularly an assay method in which a substance to be assayed in a liquid sample is quantitatively determined using an electrode and at least one oxidation-reduction enzyme, the following oxidation-reduction enzymes are used as typical examples.
(1) Oxidases
These enzymes contain a flavin coenzyme, a metal atom, a heme or the like and use oxygen as the electron acceptor in the living body.
Examples: glucose oxidase, amino-acid oxidase, lactate oxidase, urate oxidase, xanthine oxidase, ascorbate oxidase and the like.
(2) Hydroperoxidases
In the living body, they use hydrogen peroxide as the acceptor.
Examples: peroxidase, catalase and the like.
(3) Dehydrogenases
In the living body, they use AND (H) and NADP (H) as the acceptor (or donor).
Examples: alcohol dehydrogenase, lactate dehydrogenase and the like.
Of these oxidation-reduction enzymes, glucose oxidase (GOD) is used in enzyme electrodes for the measurement of blood glucose level and horseradish peroxidase (HRPO) is used as a label enzyme in specific binding assays such as DNA hybridization, immunoassay and the like. According to experiments conducted by the present inventors, both of glucose oxidase and horseradish peroxidase showed superior current response in a buffer when hydroquinone (HQ)/benzoquinone (BQ) was used as the electron mediator, in comparison with the use of conventional electron mediators such as ferrocene derivatives, inorganic ions, metal complexes and the like. However, these electron mediators were not able to show stable and reproducible response in the presence of blood, urine and the like because of the interfering substances contained therein.
Similar interference in the presence of blood, urine and the like was found also in the case of p-phenylenediamine (PPD)-derived electron mediators such as PPD, TMPD, N,N,N',N'-tetraethyl-p-phenylenediamine (TEPD) and the like which show excellent current response in a buffer solution.
In addition, the present inventors have attempted to prepare a dry chemistry type chips by impregnating a piece of cellulose filter paper or glass filter paper with a solution of HQ, PPD, TMPD or TEPD and drying the resulting piece on an electrode (by freeze-drying, vacuum drying, air-drying or the like) or arranging it on an electrode after drying so that the impregnated electron mediator can dissolve in a liquid sample at the time of measurement. This chips, however, showed a low current response and extremely poor characteristics.
Results of detailed studies conducted by the present inventors have indicated the following problems as the principal cause of such phenomena: